Nitric oxide (NO) mediated vasodilatation is one of the basic vascular responses, and the identification of NO as the endothelial derived relaxing factor was one of the fundamental advances in understanding vascular reactivity. The molecular mechanism for NO mediated smooth muscle cells relaxation is known to involve the activation of guanylate cyclase, an increase in cGMP and the subsequent activation of type I protein kinase G (PKGI). PKGI has multiple targets that lead to relaxation of smooth muscle, including phosphorylation of the maxi K+ channel to produce a hyperpolarization of the smooth muscle, a decrease in Ca2+ flux, and an activation of myosin light chain (MLC) phosphatase. MLC phosphatase is a holoenzyme] consisting of an approximately 38 kDa PPIcdelta catalytic subunit, a 20 kDa subunit of unknown function, and a myosin targeting subunit (MYPT1) of 110-130 kDa. Alternative splicing of a 31 bp 3' exon is responsible for the expression of leucine zipper positive (LZ+) or LZ MYPT1 isoforms, and it has recently been demonstrated that the C-terminal LZ of MYPT1 jis important for mediating the vascular response to NO. We have demonstrated that the sensitivity to cGMP mediated smooth muscle relaxation correlates with the relative expression of LZ+ and LZ MYPT1 isoforms, and over expression of LZ+ or LZ MYPT1 isoforms in cultured smooth muscle cells modulates cGMP mediated MLC20 dephosphorylation. However, we also demonstrated that both LZ+ and LZ MYPT1 isoforms bind to PKGI. These data suggest that 'although a MYPT1 expressing a LZ is required for cGMP mediated activation of MLC phosphatase activity, the activation of MLC phosphatase activity is independent of a LZ-LZ interaction of PKGI with MYPT1. Others have suggested that coiled-coil domains mediate the association of PKGI with MYPT1. PKGI has a coiled-coil domain at its NH2-terminus, and there are predicted coiled-coil domains of MYPT1 between aa 647-705 and aa 888-928. Thus, the hypothesis on which this application is based is that following cGMP activation of PKG, PKG and MYPT1 interact via coiled-coil domains, and PKG activates MLC phosphatase activity by a phosphorylation of MYPT1. To test this hypothesis, we will determine the sites on MYPT1 phosphorylated by PKGI during cGMP stimulation (Specific Aim 1); the sites responsible for the interaction of MYPT1 and PKGI (Specific Aim 2); and the consequences of changes in MYPT1 structure on the regulation of MLC phosphatase activity (Specific Aim 3). These specific aims are designed to determine the structure-function relation of MYPT1 for NO mediated smooth muscle relaxation. Initial experiments will use biochemical techniques to demonstrate the method by which PKGI activates MLC phosphatase activity, and then we will express the several MYPT1 truncation mutants and MYPT1 bearing mutations at critical sites in cultured smooth muscle cells to determine the effect of these changes on the regulation of MLC phosphatase activity, and cGMP mediated smooth muscle relaxation. Thus the results will determine the molecular mechanism by which cGMP activates! MLC phosphatase activity to produce smooth muscle relaxation.